Understanding the human microbiome is critical in maintaining human health and preventing disease, but it has been unclear how specific microbes affect health and disease because the majority of microbes cannot be cultivated using traditional methods. Technologies are needed that can both increase the success rate for cultivating microbes and target cultivation efforts towards microbes of high biomedical interest. This project will use microfluidic confinement to overcome the limitations of traditional cultivation and targeting methods by developing "single cell confinement technology". Stochastic confinement of single cells in droplets of small volumes (picoliters to nanoliters) will isolate microbial species and, potentially, enable cultivation of new microbes by initiating high-density growth from a single cell. The droplets created by this single cell confinement technology can be split to perform multiple assays in parallel on clonal sister populations, enabling killing assays to be performed to identify microbes in one sister population, and using of the other sister population for growth. In this technology, to target cultivation efforts towards microbes of biomedical interest, new species will be identified via two complementary approaches: gene-based assays and function-based assays. Gene-based assays, informed by existing metagenomic data, will identify desired functional genes and 16S sequences, and function-based assays will identify desired functions even if they are not associated with a known gene sequence. The identified microbial species will then be targeted for scale-up of microcolonies to make them available for sequencing and further study. We will develop and validate the single cell confinement technology by using sulfur-reducing bacteria from the human colon as the test system. Sulfur-reducing bacteria are of high biomedical importance, associated with Ulcerative Colitis and intra-abdominal infections, but are still poorly understood. We will first use a model consortium of gut-derived microorganisms, containing a representative sulfur-reducing bacterium Bilophila wadsworthia, to develop and optimize the technology. Next, we will develop gene-based and function- based assays and test them by identifying sulfate reducing bacteria in model mixtures. Finally, we will use these cultivation approaches and assays to cultivate and select new sulfur-reducing bacteria from the human colon. This technology will be generally applicable to identify and cultivate all classes of microbes in the human gut microbiome. This project will impact biomedical science and public health by developing and validating technologies for increasing our understanding of the relationship between genes and functions in the human gut microbiome, and therefore microbial contributions to both health and disease. PUBLIC HEALTH RELEVANCE: Statement Microbes are critical to the function of the gastrointestinal tract. Understanding their role in human health and disease requires cultivation, but the majority of the species in the human gut microbiome are difficult to cultivate. This application will develop confined-based technology to enhance cultivation of microbes from human colon and target the cultivation efforts by using complementary assays to identify microbes with genes and functions of high biomedical interest.